Isoprene synthase and method of preparing isoprene using thereof

ABSTRACT

Provided are a novel isoprene synthase derived from sweet potato and a method of preparing isoprene using the same, and more specifically, a novel isoprene synthase derived from sweet potato, a gene encoding the isoprene synthase, a host cell transformed with the gene, and a method of preparing isoprene using the same. The isoprene synthase of the present invention may have higher isoprene productivity as compared to isoprene synthases known in the related art to thereby be effectively used in isoprene biosynthesis and preparation of an isoprene polymer using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of International Application No. PCT/KR2015/007851 filed Jul. 28, 2015, and claims priority to Korean Patent Application No. 10-2014-0095972 filed Jul. 28, 2014, the disclosures of which are hereby incorporated in their entirety by reference.

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 1700617_ST25.txt. The size of the text file is 167,544 bytes, and the text file was created on Jan. 26, 2017.

TECHNICAL FIELD

The present invention relates to a novel isoprene synthase and a method of preparing isoprene using the same, and more specifically, to a polynucleotide encoding the novel isoprene synthase, a recombinant host cell having the polynucleotide introduced, and a method of preparing isoprene using the same.

BACKGROUND ART

Isoprenoids are isoprene polymers that find use in pharmaceuticals, neutraceuticals, flavors, fragrances, and rubber products. Supplies of natural isoprenoid, however, are restricted due to ecological concerns. For this reason, and in order to provide isoprenoid compositions having less impurities and greater uniformity, isoprenoids such as rubber are often produced synthetically. Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohol. Commercially viable quantities of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or by dehydration of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4^(th) ed., Wiley-VCH, pp. 117-122, 2003). The C5 skeleton can also be synthesized from smaller subunits.

It would be desirable, however, to have a commercially viable method of producing isoprene that was independent of nonrenewable resources. Biosynthetic production of isoprene occurs by two distinct metabolic pathways (Julsing et al., Appl Microbiol Biotechnol, 75:1377-1384, 2007). In eukaryotes and archae, isoprene is formed via the mevalonate (MVA) pathway, while some eubacteria and higher plants produce isoprene via the methylerythritol phosphate (MEP) pathway. Isoprene emissions from plants are light and temperature-dependent and increase with the association to leaf development.

An isoprene-producing enzyme, isoprene synthase, has been identified in Aspen trees (Silver and Fall, Plant Physiol, 97:1588-1591, 1991; and Silver and Fall, J Biol Chem, 270:13010-13016, 1995) and is believed to be responsible for the in vivo production of isoprene from whole leaves. Bacterial production of isoprene has also been described (Kuzma et al., Curr Microbiol, 30:97-103, 1995; and Wilkins, Chemosphere, 32:1427-1434, 1996), and it varies in amount according to the phase of bacterial growth and the nutrient content of the culture medium (U.S. Pat. No. 5,849,970 to Fall et al.; and Wagner et al., J Bacteriol, 181:4700-4703, 1999).

The levels of isoprene obtainable through bacterial systems of the prior art, however, are insufficient for commercial uses. Thus, what the art needs is an effective and large scaled bacterial or microbial isoprene production process to provide feedstock for the manufacture of isoprene.

Accordingly, as a result of an effort for developing a method of preparing isoprene using a novel isoprene synthase gene having excellent isoprene productivity, the present inventors performed mining on a novel isoprene synthase gene, and confirmed that a recombinant microorganism transformed with the isoprene synthase gene has more excellent isoprene productivity than that of a host cell transformed with the isoprene synthase gene known in the art, thereby completing the present invention.

SUMMARY OF THE INVENTION Technical Problem

An object of the present invention is to provide an isoprene synthase having excellent isoprene productivity and a gene encoding the isoprene synthase.

Another object of the present invention is to provide a recombinant host cell expressing an isoprene synthase and a method of preparing isoprene by culturing the recombinant host cell.

Solution to Problem

In order to achieve the foregoing objects, the present invention provides an isoprene synthase comprising an amino acid sequence of SEQ ID NO: 1; or an amino acid sequence having 70% or more sequence homology to the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a polynucleotide encoding the isoprene synthase as described above and a recombinant vector into which the polynucleotide is operably introduced.

Further, the present invention provides a recombinant host cell into which the polynucleotide or the recombinant vector as described above is introduced.

In addition, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 3 optimized by codon optimization of the polynucleotide sequence encoding the isoprene synthase for blue-green algae (cyanobacteria); and a recombinant vector into which the polynucleotide is operably introduced.

In addition, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 33 optimized by codon optimization of the polynucleotide sequence encoding the isoprene synthase for yeast; and a recombinant vector into which the polynucleotide is operably introduced.

Further, the present invention provides recombinant blue-green alga (cyanobacteria) or recombinant filamentous fungi; into which the polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 3 as described above or the recombinant vector comprising the polynucleotide as described above is introduced.

Further, the present invention provides recombinant yeast; into which the polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 33 as described above or the recombinant vector comprising the polynucleotide as described above is introduced.

In addition, the present invention provides a method of preparing isoprene including: (a) culturing the recombinant host cell as described above to prepare isoprene; and (b) obtaining the prepared isoprene.

Further, the present invention provides a method of preparing isoprene including: (a) culturing the recombinant blue-green algae (cyanobacteria), filamentous fungi or yeast as described above to prepare isoprene; and (b) obtaining the prepared isoprene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a and FIG. 1b show an amino acid sequence alignment result of l. batatas-derived isoprene synthase with known isoprene synthases (SEQ ID NOS: 37-51).

FIG. 2 is directed to multiple sequence alignment (MSA) result showing the sequence around the substrate binding amino acids in isoprene synthase (SEQ ID NOS: 52-66).

FIG. 3 shows a multiple sequence alignment (MSA) result to around the substrate-binding amino acid sequence (SEQ ID NOS: 67-96).

FIG. 4 shows a phylogenetic tree among isoprene synthase candidates.

FIG. 5 shows a multiple sequence alignment (MSA) result of the isoprene synthase candidates.

FIG. 6 shows a map of pETDuet-1 vector.

FIG. 7 shows an expression of an isoprene synthase of isoprene synthase clone comprising a pBAT4 construct (trc promoter) of P. alba- and I. batatas-derived isoprene synthase genes.

FIG. 8 shows isoprene productivity of the pBAT4 construct (trc promoter) comprising P. alba- and I. batatas-derived isoprene synthase genes.

FIG. 9 shows an expression of an isoprene synthase of an E. coli strain transformed into a pETDuet-1 vector having five kinds of isoprene synthases (I. batatas, E. photiniifolius, P. alba, P. montana and A. hypogaea) introduced thereinto.

FIG. 10 shows isoprene productivity of an E. coli strain transformed into a pETDuet-1 vector, in which five kinds of isoprene synthases (I. batatas, E. photiniifolius, P. alba, P. montana and A. hypogaea) are inserted.

FIG. 11 shows inhibition in growth of an E. coli strain transformed into a pETDuet-1 vector, in which five kinds of isoprene synthases (I. batatas, E. photiniifolius, P. alba, P. montana and A. hypogaea) are inserted.

FIG. 12 shows time dependent isoprene productivity after expression of isoprene synthases in an E. coli strain transformed into a pETDuet-1 vector in which I. batatas of isoprene synthases and P. alba of isoprene synthases, respectably, are inserted.

FIG. 13 shows time dependent growth inhibition in of an E. coli strain transformed into a pETDuet-1 vector, in which I. batatas of isoprene synthases and P. alba of isoprene synthases, respectably, are inserted.

FIG. 14 shows plasmid for expression of IspS, IDI and HMG-CoA reductase in S. cerevisiae.

FIG. 15 shows plasmid for expression of IspS, and IDI in P. kudriavzevii.

FIG. 16 shows plasmids for expression of HMG-reductase in P. kudriavzevii.

FIG. 17 shows plasmid for expression of IspS in filamentous fungi.

FIG. 18 shows isoprene production by P. kudriavzevii transformants in YP medium containing 1% glucose (grey) or 1% glycerol (black) as the carbon source.

FIG. 19 shows plasmid pCIL105 for expression of I. batatas IspS in T. reesei.

FIG. 20 shows PCR analysis for the presence of the IspS expression cassette in T. reesei transformed with pCIL105. Wild type strain QM6a, parent and DDIW are negative controls.

DESCRIPTION OF THE INVENTION

Unless defined otherwise, all the technical and scientific terms used herein have the same meanings as those generally understood by persons skilled in the art to which the present invention pertains. Generally, the nomenclature used herein are well known and commonly employed in the art.

In a first aspect of the present invention, the present invention provides an isoprene synthase comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, still more preferably at least 90%, still more preferably at least 95%, more and more preferably at least 98%, most preferably at least 99% sequence homology to the amino acid sequence of SEQ ID NO: 1.

The isoprene synthase belongs to a terpene synthase (TPS)-B family, and has an amino acid sequence specifically conserved in isoprene synthase, referred to as an “isoprene score” (Sharkey et al., Evolution, 67:1026, 2013). Important amino acids in the isoprene synthase are F338, S445, F485 and N505 based on amino acid sequences of the Populus alba- and Populus canescens-derived isoprene synthases, and phenylalanine of F338 and F485 are important amino acids to decrease the size of a substrate-binding site so that large substrates such as geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, and the like, are not allowed to enter into an active site of an enzyme.

Therefore, the isoprene synthase of the present invention is characterized by comprising an amino acid sequence having 90% or more sequence homology to sequences at positions corresponding to RDRLMESFFW at position Nos. 257-266, FKLVTVLDDVYD at position Nos. 287-298, F369, SVS at position Nos. 394-396, FRLANDLSSSKAEIERGETANSI at position Nos. 433-455, and YQYGDAH at position Nos. 515-521 in an amino acid sequence of SEQ ID NO: 1. In addition, the isoprene synthase of the present invention is characterized by having at positions corresponding to the positions in SEQ ID NO: 1 at least one, preferably at least two, more preferably at least three, still more preferably at least four of the amino acids W266, F287, F369, F433, N453 and Y515.

The isoprene synthase of SEQ ID NO: 1 of the present invention comprises an amino acid sequence derived from sweet potato (Ipomoea batatas).

An amino acid sequence alignment result of I. batatas-derived isoprene synthase is shown in FIG. 1a and FIG. 1b , and an amino acid sequence alignment result of isoprene synthase candidates including P. alba-, A. hypogaea-derived isoprene synthases which are two known isoprene synthases, as a reference is shown in FIG. 2.

As a result of searching patent sequence database based on I. batatas-derived isoprene synthase having an amino acid sequence of SEQ ID NO: 1 according to the present invention, it was confirmed that the isoprene synthase derived from sweet potato of the present invention has 56% sequence homology to Quercus petraea-derived isoprene synthase (US Patent Application Publication No. 2013/0330709) and has low sequence homology to the isoprene synthase known in the related art.

In a second aspect of the present invention, the present invention provides a polynucleotide encoding the isoprene synthase.

In the present invention, the polynucleotide may be codon-optimized for microorganisms selected from the group consisting of E. coli, bacillus genus strain, blue-green algae (cyanobacteria), yeast and filamentous fungi. Also the introns may be removed from the original gene. In the present invention the term “polynucleotide” is used to mean the gene in its original or modified form or the coding sequence in its original or modified form.

The codon optimization in the present invention means that a codon having an average codon frequency less than 12% on the microorganism is substituted with a codon having an average codon frequency more than 12% on the microorganism.

In an exemplary embodiment of the present invention, with a target of a isoprene synthase polynucleotide of SEQ ID NO: 2, the codon optimization was performed to enable expression in either Synechocystis sp. (strain PCC6803), which is blue-green algae cyanobacteria, or E. coli (SEQ ID NO: 3). This same codon optimized nucleotide sequence for bacteria can be used also for filamentous fungi. In an exemplary embodiment of the present invention SEQ ID NO:3 was used for the filamentous fungus Trichoderma.

In another exemplary embodiment of the present invention, with a target of an isoprene synthase polynucleotide of SEQ ID NO: 2, the codon usage was optimized for S. cerevisiae (SEQ ID NO:33) to enable expression either in S. cerevisiae or Pichia (P. kudriavzevii).

Therefore, in a preferred embodiment of the present disclosure, the polynucleotide encoding the isoprene synthase might comprise any one nucleotide sequence of SEQ ID NOs: 2, 3 or 33, but the present invention is not limited thereto.

In a third aspect of the present invention, the present invention provides a polynucleotide encoding an isoprene synthase comprising a nucleotide sequence of SEQ ID NO: 3 by codon optimization of the polynucleotide encoding the isoprene synthase for blue-green algae (cyanobacteria), and E. coli, useful also for other bacteria and filamentous fungi, in particular Trichoderma and Aspergillus.

In a fourth aspect of the present invention, the present invention provides a polynucleotide encoding an isoprene synthase comprising a nucleotide sequence of SEQ ID NO: 33 by codon optimization of the polynucleotide encoding the isoprene synthase for Saccharomyces cerevisiae, useful also for Saccharomyces, Pichia and other yeasts.

In a fifth aspect of the present invention, the present invention provides a recombinant vector into which the polynucleotide is operably introduced; and a recombinant host cell into which the polynucleotide or the recombinant vector is introduced.

The recombinant vector in the present invention may contain a promoter for expressing the polynucleotide.

Examples of the promoter contained in the recombinant vector could include psbA2, trc, rbcL, petJ, psaA, psaB, tac, cpcB, petC or lac promoters for expression in blue-green algae (cyanobacteria), PGK1, TPI1, TDH, PDC1, FBA1, ENO1, ENO2, PYK1, ADH1, or TEF1 promoters for expression in yeast, preferably the promoter is selected from the group consisting of PGK1, TPI1 and TEF1 for expressing in yeast, cbh1, gpdA, glaA, pdc or exlA promoters for expression in filamentous fungi, and a CMV35S promoter for expression in a plant cell, but the present invention is not limited thereto.

In a preferred embodiment of the present invention, a polynucleotide sequence is contained in plasmids. In another preferred embodiment of the present invention, a polynucleotide sequence is incorporated into the genome of a host cell. In some preferred embodiments of the present invention, a host is selected from the group consisting of gram-positive bacterial cells, gram-negative bacterial cells, filamentous fungal cells, and yeast cells, but the present invention is not limited thereto.

In some preferred embodiments of the present invention, escherichia species (E. coli), pantoea species (Pantoea citrea), bacillus species (Bacillus subtilis), yarrowia species (Yarrowia lipolytica), and trichoderma species (Trichoderma reesei), but the present invention is not limited thereto. In some preferred embodiments, the host cell is cultured in a medium containing carbon sources selected from the group consisting of CO₂, bicarbonate, glucose, glycerol, glycerine, dihydroxyacetone, yeast extract, biomass, molasses, sucrose, galactose, sorbose, sorbitol, xylose, arabinose, cellulose, xylan, lactose and oil, but not limited thereto.

Preferably, the host cell of the present invention may be blue-green algae (cyanobacteria), yeast, filamentous fungi, or plant cells.

In a preferred embodiment of the present invention, the filamentous fungi may be Trichoderma genus, Mucor genus, Mortierella genus, Neurospora genus or Aspergillus genus, advantageously the filamentous fungi is Trichoderma or Aspergillus genus and the plant cell may be Nicotiana genus, Catharantus genus or Hyroscyamus genus plant cells.

In a sixth aspect of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 3 by codon optimization of the polynucleotide encoding the isoprene synthase on blue-green algae (cyanobacteria), useful also for other bacteria and filamentous fungi, and a recombinant vector into which the polynucleotide is operably introduced; and a recombinant blue-green algae (cyanobacteria), bacteria or filamentous fungi into which the polynucleotide or the recombinant vector is introduced.

In a seventh aspect of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 33 by codon optimization of the polynucleotide encoding the isoprene synthase on Saccharomyces cerevisiae useful for Saccharomyces, Pichia and other yeasts, and a recombinant vector into which the polynucleotide is operably introduced; and a recombinant yeast into which the polynucleotide or the recombinant vector is introduced.

In the present invention, the recombinant vector may comprise psbA2, trc, rbcL, petJ, psaA, psaB, tac, cpcB, petC or lac promoter for expression in blue-green algae (cyanobacteria).

Preferably, the blue-green algae may be unicellular blue-green algae or multicellular blue-green algae. The unicellular blue-green algae may be Synechocystis genus or Synechococcus genus strain, and the multicellular blue-green algae may be Gloeocapsa genus or filamentous cyanobacteria.

In a preferred embodiment of the present invention, the filamentous cyanobacteria may be Nostoc genus, Anabaena genus or Arthospira genus, and the yeast may be Saccharomyces genus, Pichia genus, Candida genus, kazachstania genus, Kluyveromyces genus, Hansenula genus, Rhodosporidium genus, Cryptococcus genus or Yarrowia genus.

The polynucleotide is operably linked when being disposed with other nucleic acid sequences in a functional relationship. This may be polynucleotide and a regulatory sequence(s) linked in the manner of enabling polynucleotide expression when appropriate molecules (for example, transcriptional activation protein) are bound to the regulatory sequence(s). For example, nucleotide sequence for a pre-sequence or a secretion leader is operably linked to nucleotide sequence for a polypeptide when being expressed as a pre-protein participating in secretion of polypeptide; a promoter or an enhancer is operably linked to a coding sequence when having an influence on transcription of sequence; or a ribosome binding site is operably linked to a coding sequence when having an influence on transcription of sequence; or a ribosome binding site is operably linked to a coding sequence when being disposed so that translation is easily performed. In general, term: ‘operably linked’ means that the linked nucleotide sequences are contacted with each other, or in the case of the secretion leader, contacted with the nucleotide sequence and present within a leading frame. However, the enhancer does not need to have a contact. The link of the sequences is performed by ligation (linkage) in a convenient restriction enzyme site. When the site does not exist, a synthetic oligonucleotide adaptor or a linker according to general method is used.

A method of inserting the polynucleotide into the genome of a host cell may be a generally known genetic engineering method, and a non-viral transfer method may include electroporation, lipofection, microinjection, biolistic, virosome, liposome, immuno-liposome, multivalent cation or lipid:nucleic acid conjugate, naked DNA, artificial virion and chemical-enhanced uptake of DNA. Sonoporation, for example, a method using a sonitron 2000 system (Rich-Mar) may be used for transfer of nucleic acids, and other representative nucleic acid transfer systems include Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molesular Syetem (Holliston, Mass.). The lipofection method is disclosed in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagent is commercially available, for example, Transfectam™ and Lipofectin™. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner (WO 91/17424 and WO 91/16024), and may be transferred to cells via ex-vivo administration or to target tissues via in-vivo administration. A method of preparing lipid:nucleic acid complex, containing targeted liposomes such as immunolipid complexes, is well known in the art (Crystal, Science., 270:404-410, 1995; Blaese et al., Cancer Gene Ther., 2:291-297, 1995; Behr et al., Bioconjugate Chem., 5:382389, 1994; Remy et al., Bioconjugate Chem., 5:647-654, 1994; Gao et al., Gene Therapy., 2:710-722, 1995; Ahmad et al., Cancer Res., 52:4817-4820, 1992; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; 4,946,787).

The tropism of a retrovirus may be altered by unification with foreign envelope proteins and thereby expand kinds of target cells. A lentiviral vector is a type of retroviral vector that is able to transduce or infect non-dividing cells and produce high viral titers. A retroviral gene transfer system is determined depends on the target tissue. The retroviral vector includes cis-acting long terminal repeats with packaging capacity for 6-10 kb of foreign sequence. The minimum cis-acting LTRs which are sufficient for replication and packaging of the vectors may be used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (Buchscher et al., J. Virol., 66:2731-2739, 1992; Johann et al., J. Virol., 66:1635-1640, 1992; Sommerfelt et al., Virol., 176:58-59, 1990; Wilson et al., J. Virol., 63:2374-2378, 1989; Miller et al., J. Virol., 65:2220-2224, 1991).

In a case of temporarily expressing a sucrose phosphorylase protein, an adenoviral based system may be frequently used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. When using the vectors, high titer and high levels of expression may be obtained and a mass-production is possible with a relatively simple system. In addition, Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, for example, for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy (West et al., Virology., 160:38-47, 1987; U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy., 5:793-801, 1994; Muzyczka, J. Clin. Invest., 94:1351, 1994), and a construction of recombinant AAV vectors were already known (U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol., 5:3251-3260, 1985; Tratschin, et al., Mol. Cell. Biol., 4:20722081, 1984; Hermonat & Muzyczka, PNAS., 81:6466-6470, 1984; Samulski et al., J Virol., 63:3822-3828, 1989). In clinical trials, at least six viral vector approaches are currently available for gene transfer, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood., 85:3048, 1995; Kohn et al., Nat. Med., 1:1017, 1995; Malech et al., PNAS., 94:12133, 1997), and PA317/pLASN is the first therapeutic vector used in a gene therapy trial (Blaese et al., Science., 270:475-480, 1995), and transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors (Ellem et al., Immunol Immunother., 44(1):10-20, 1997; Dranoff et al., Hum. Gene Ther., 1:111-2, 1997).

Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al., Lancet., 351:9117, 1998; Kearns et al., Gene Ther., 9:748-55, 1996).

In an eighth aspect of the present invention, the present invention provides a method of preparing isoprene including: (a) culturing the recombinant host cell as described above to prepare isoprene; and (b) obtaining the prepared isoprene.

The culturing of the step (a) may be performed in a medium containing carbon sources selected from the group consisting of CO₂, bicarbonate, glucose, glycerol, glycerine, dihydroxyacetone, yeast extract, biomass, molasses, sucrose, galactose, sorbose, sorbitol, xylose, arabinose, cellulose, xylan, lactose and oil.

In a ninth aspect of the present invention, the present invention provides a method of preparing isoprene including: (a) culturing the recombinant blue-green algae (cyanobacteria), bacteria, recombinant filamentous fungi or yeast as described above to prepare isoprene; and (b) obtaining the prepared isoprene.

The methods may additionally include (c) polymerizing isoprene.

Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Second Edition, Cold Spring Harbor, 1989; and Ausubel et al., “Current Protocols in Molecular Biology,” 1987).

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provides those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

In combination with isoprene synthase expression of other genes contributing to isoprene production can be introduced into blue green algae (cyanobacteria), bacteria, filamentous fungi or yeast, cells. For example, the idi gene coding for isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) can be introduced to enhance conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) that is the substrate of isoprene synthase. Also one or more genes coding for the mevalonate pathway components, mevalonate kinase (EC2.7.1.36), phosphomevalonate kinase (EC2.7.4.2), pyrophoshomevalonate decarboxylase (EC 4.1.1.33), acetoacetyl-CoA thiolase (EC2.3.1.9), HMG-CoA synthase (EC2.3.3.10) or HMG-CoA reductase (EC1.1.1.34) can be cloned under a suitable cyanobacteria, bacteria, filamentous fungi or yeast promoter that allow expression in these hosts. Two or more genes are expressed as a transcriptional fusion under the control or under the transcriptional control of a cyanobacteria or bacteria promoter. When two or more genes are expressed in, filamentous fungi or yeast, each gene is expressed under the control of an individual filamentous fungal or yeast promoter. Advantageous for expression of isoprene synthase is for example a combination of IDI and HMG reductase in filamentous fungi and in yeast, in particular in Pichia and Saccharomyces.

As used herein, the term 2-methyl-1,3-butadiene (CAS #78-79-5) (“isoprene”) refers to the direct and final volatile C5 hydrocarbon product from the elimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate (DMAPP), and does not involve the linking or polymerization of [an] IPP molecule(s) to [a] DMAPP molecule(s).

As used herein, the terms “isoprene synthase,” and “IspS,” refer to the enzymes that catalyze the elimination or pyrophosphate from diemethylallyl diphosphate (DMAPP) to form isoprene.

In some embodiments, the term “IspS” refers to a naturally occurring mature enzyme or portion thereof.

The present invention comprises proteins comprising an amino acid sequence having 70% or more identity with the amino acid sequence of Isoprene synthase from sweet potato, the proteins comprise “variant proteins”.

In some preferred embodiments, variant proteins differ from a parent protein (e.g., set forth as SEQ ID NO:1) by a small number of amino acid residues. The number of differing amino acid residues may be one or more, preferably 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In some preferred embodiments, the number of different amino acids between variants is between 1 and 10. In some particularly preferred embodiments, related proteins and particularly variant proteins comprise at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, a related protein or a variant protein as used herein refers to a protein that differs from another related protein or a parent protein in the number of prominent regions. For example, in some embodiments, variant proteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions that differ from the parent protein.

In present invention, the proteins comprising an amino acid sequence having 70% identity with amino acid sequence set forth as SEQ ID NO:1 of Isoprene synthase can be generated with several methods including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNA segment) that encodes a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons). Polynucleotides introduced into host cells may comprise the coding region without introns and with or without the preceding or following regions of the original gene. Furthermore the codons may be modified to be more suitable for the host cell.

As used herein, “homology” refers to sequence similarity or identity, with identity being preferred. This homology is determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv Appl Math, 2:482, 1981; Needleman and Wunsch, J Mol Biol, 48:443, 1970; Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.; and Devereux et al., Nucl Acid Res, 12:387-395, 1984).

As used herein, an “analogous sequence” is one wherein the function of the polynucleotide is essentially the same as the polynucleotide based on sweet potato isoprene synthase (IspS). Additionally, analogous polynucleotides include at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the batate isoprene synthase. In additional embodiments more than one of the above properties applies to the sequence. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences.

Thus, “percent (%) nucleic acid sequence identity” is defined as the percentage of nucleotide residues in a candidate sequence that is identical to the nucleotide residues of the starting sequence (i.e., the sequence of interest). A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells expresses genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. “Recombination,” “recombining,” and generating a “recombined” nucleic acid are generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric gene.

As used herein, the terms “amplification” and “gene amplification” refer to a process by which specific polynucleotides are disproportionately replicated such that the amplified polynucleotide becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and “amplification vector” refer to a polynucleotide or a vector comprising a polynucleotide, which permits the amplification of that polynucleotide under appropriate growth conditions.

“Homologous sequences” as used herein means a nucleic acid or polypeptide sequence having 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 88%, 85%, 80%, 75%, or 70% sequence identity to another nucleic acid or polypeptide sequence when optimally aligned for comparison. In some embodiments, homologous sequences have between 85% and 100% sequence identity, while in other embodiments there is between 90% and 100% sequence identity, and in more preferred embodiments, there is 95% and 100% sequence identity.

As used herein “amino acid” refers to peptide or protein sequences or portions thereof. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably.

As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell. Examples of heterologous proteins include enzymes such as isoprene synthases. In some embodiments, the polynucleotides encoding the proteins are naturally occurring genes, while in other embodiments mutated and/or synthetic polynucleotides are used.

As used herein, “homologous protein” refers to a protein or polypeptide native or naturally occurring in a cell. In preferred embodiments, the cell is a Gram-negative cell, while in particularly preferred embodiments the cell is an Escherichia host cell.

An enzyme is “overexpressed” in a host cell if the enzyme is expressed in the cell at a higher level that the level at which it is expressed in a corresponding wild-type cell.

The terms “protein” and “polypeptide” are used interchangeability herein. The 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used through out this disclosure. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

The term “mature” form of a protein or peptide refers to the final functional form of the protein or peptide. To exemplify, a mature form of sweet potato isoprene synthase includes the amino acid sequence of SEQ ID NO:1.

The term “precursor” form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a “signal sequence” operably linked, to the amino terminus of the prosequence. The precursor may also have additional polynucleotides that are involved in post-translational activity (e.g., polynucleotides cleaved therefrom to leave the mature form of a protein or peptide).

“Naturally occurring enzyme” refers to an enzyme having the unmodified amino acid sequence identical to that found in nature. Naturally occurring enzymes include native enzymes, those enzymes naturally expressed or found in the particular microorganism.

The term “identical” in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using one of the following sequence comparison or analysis algorithms.

The term “optimal alignment” refers to the alignment giving the highest percent identity score. “Percent sequence identity,” “percent amino acid sequence identity,” “percent gene sequence identity,” and/or “percent nucleic acid/polynucleotide sequence identity,” with respect to two amino acid, polynucleotide and/or gene sequences (as appropriate), refer to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical.

The phrase “substantially identical” in the context of two nucleic acids or polypeptides thus refers to a polynucleotide or polypeptide that comprising at least 70% sequence identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferably at least 99% sequence identity as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

As used herein, “corresponding to,” refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, “corresponding region,” generally refers to an analogous position along related proteins or a parent protein.

As used herein, the terms “multiple sequence alignment” and “MSA” refer to the sequences of multiple homologs of a starting gene that are aligned using an algorithm (e.g., Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence” refer to an archetypical amino acid sequence against which all variants of a particular protein or sequence of interest are compared. The terms also refer to a sequence that sets forth the nucleotides that are most often present in a DNA sequence of interest. For each position of a gene, the consensus sequence gives the amino acid that is most abundant in that position in the MSA.

As used herein, the term “consensus mutation” refers to a difference in the sequence of a starting gene and a consensus sequence. Consensus mutations are identified by comparing the sequences of the starting gene and the consensus sequence obtained from a MSA. In some embodiments, consensus mutations are introduced into the starting gene such that it becomes more similar to the consensus sequence. Consensus mutations also include amino acid changes that change an amino acid in a starting gene to an amino acid that is more frequently found in an MSA at that position relative to the frequency of that amino acid in the starting gene. Thus, the term consensus mutation comprises all single amino acid changes that replace an amino acid of the starting gene with an amino acid that is more abundant than the amino acid in the MSA.

As used herein, the term “headspace” refers to the vapor/air mixture trapped above a solid or liquid sample in a sealed vessel.

Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.

Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise.

EXAMPLES

Hereinafter, the present invention will be described in detail with reference to the following Examples. However, the following examples are only for exemplifying the present invention and it will be obvious to those skilled in the art that the scope of the present invention is not construed to be limited to these examples.

Example 1 Genome Mining for Novel Isoprene Synthase Genetic Search

The isoprene synthase belongs to a terpene synthase (TPS)-B family, and has an amino acid specifically conserved in isoprene synthase, referred to as an “isoprene score” (Sharkey et al., Evolution, 67: 1026, 2013). Important amino acids in the isoprene synthase are F338, S445, F485 and N505 based on amino acid numbering of sequences of the Populus alba- and Populus canescens-derived isoprene synthases, and phenylalanines of F338 and F485 respectively are amino acids which have an important role to decrease a size of a substrate-binding site so that large substrates such as geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, and the like, are not allowed to enter into an active site of an enzyme.

Bi-functional myrcene synthase of Humulus lupulus having low activity as compared to other isoprene synthases lacks F485. N505 has a critical role to determine an ion requirement of terpene synthases, and terpene synthase without an ion requirement has cationic lysine, serine, or asparagine positioned at position No. 505. S445 is present at a first position of triple serine motif, and other TPS-b protein usually has valine (Val) and isoleucine (Ile) in the middle of the triple serine motif; however, S445 present at the first position of the triple serine motif is conserved in almost all Tps-b family and thus, it is judged that S446 positioned in the middle of the triple serine motif is not important in preparation of isoprene.

In addition, W317 and Y565, which are amino acids present in almost all TPS family sequences, are essential to define a size of a substrate-bound pocket.

In addition, N489 is conserved in isoprene/ocimene clade of the Tps-B

In order to determine isoprene Synthase candidates, a homology-based database search was conducted. Isoprene synthase sequences using Query sequence are as follows:

1) Populus alba (white poplar, Uniport: Q50L36), Populus canescens (grey poplar, Uniprot: Q9AR86, PDB: 3N0G) and Pueraria montana var. lobata (kudzu wine, Uniprot: Q6EJ97);

2) Arachis hypogaea (peanut, SEQ ID No: 3), Glycine max (soybean, SEQ ID No:5 and SEQ ID No:7), Mucuna pruriens (velvet bean, SEQ ID No:9), Cajanus cajans (pigeon pea, SEQ ID No:11) and Quercus petraea (oak, SEQ ID No:13) derived from WO 2013/166,320A1;

3) Wisteria sp. (GenBank: AEK70969), Robinia pseudoacacia (GenBank: AEK70968), Melaleuca alternifolia (GenBank: AAP40638), Eucalyptus globulus (GenBank: BAF02831), Salix sp. (GenBank: AEK70970) and a myrcene synthase from Humulus lupulus (GenBank: ACI32638) derived from Sharkey list (Sharkey et al., Evolution, 67: 1026, 2013); and

4) 2-methyl-3-buten-2-ol (MBO) synthase (GenBank: AEB53064)(Gray et al., J Biol Chem, 286: 20582, 2011) derived from Pinus sabiniana.

In addition, several sequences derived from poplar genus were included as a reference.

The homology-based search was performed in GenBank protein databases (nr, pat and env_nr) using Uniport (SwissProt and TrEMBL) and blastp, and was performed in GenBank nucleotide databases (tsa_nt, env_nt and pat) using tblastn, and extracted when E-value is less than 1e-30.

Additionally, Uniprot/SwisProt sequences with InterPro domain annotation of “Terpene synthase, metal binding domain” protein family (PFAM: PF03936, Interpro: IPR005630) were retrieved.

The searched nucleotide sequence was translated into an amino acid sequence by GeneWise program.

tsa_nt (GenBank TSA1) which is a database including cDNA sequence through Transcriptomic study was included in Query database, and in order to increase a usable coverage of a plant genome data, transcriptomic data on plant genomes of which sequences are determined up to date was included.

Accordingly, total 9123 sequences were searched, wherein 278 sequences were searched in Uniprot/SwissProt, 1,989 sequences were searched in Uniprot/TrEMBL, 3,953 sequences were searched in GenBank nucleotide databases, and 2,905 sequences were searched in GenBank protein databases. In order to remove the repeated sequences, sequences having 80% homology with each other clustered.

In order to confirm functional diversity among the searched terpene synthases, multiple sequence alignment (MAS) and phylogenetic tree were made by using script in a unix environment. Sequences were aligned with respect to PFAM domain (PF03936) of a protein family and phylogenetic tree was made based on the MSA using FASTTREE program.

The active site of the isoprene synthase determined based on the structure of Populus canescens IpsS (PDB:3n0g) was marked to the MSA. The sequences were aligned by ClustalW, and a conserved phylogenetic tree was constructed with resampling using Genious tree builder.

As the final candidates of the plant-derived sequences, Ipomoea batatas-derived sequence and Elaeocarpus photiniifolius-derived sequence were determined.

Ipomoea batatas sequence has the key amino acids of isoprene synthases W317 (W266 in I. batatas), F338 (F287 in I. batatas), S445 (S394 in I. batatas), F485 (F433 in I. batatas), N505 (N453 in I. batatas) and Y565 (Y515 in I. batatas)

Multiple sequence alignment of I. batatas isoprene synthase sequence together with the reference sequences is shown in FIG. 1a and FIG. 1b . Alignment of the substrate binding amino acid positions is shown in FIG. 2.

Among the additional new candidates, Medicago sativa, Fragaria vesca subsp. Vesca, Morus notabilis, Populus trichocarpa, Dahlia pinnata, Sesamum indicum and Eucalyptus grandis lacked an amino acid at F338 position (amino acid numbering based on Populus alba isoprene synthase), and Mangifera indica-derived sequence had an important amino acid at F338, which seemed to have a function of the isoprene synthase.

A multiple sequence alignment (MSA) result to an active site sequence of the isoprene synthase candidates was shown in FIG. 3. Reference isoprene synthase sequences, and four sequences (tricyclene or beta-ocimene synthase) other than the reference isoprene synthases were also included as a reference, and identity % of the sequences was shown in FIG. 5.

In FIG. 5, the reference isoprene synthases were shown in bold black text, and sequences similar to the reference isoprene synthase were shown in black text, and the candidates known in the related art were shown in black text in italics. Isoprene synthase candidates (Ipomoea batatas and Elaeocarpus photiniifolius) having 4 points of isoprene scores were shown in white text with black background. Additional new candidate sequences were shown in black text with grey background, and candidates having F388 and 3 points of isoprene score were shown in white text with dark grey background. Sequences with confirmed other functions are shown in grey. The phylogenetic tree was provided with EC number, the biological name, and Blast E-values, and was visualized by Geneious software (FIG. 4).

Example 2 Cloning of Selected Isoprene Synthase Gene Candidate

Since Ipomoea batatas- and Elaeocarpus photiniifolius-derived nucleotide sequences selected in Example 1 above have a low homology to the known isoprene synthase encoding nucleotide sequences, the polynucleotides were cloned to measure an enzyme activity of protein to be expressed by the polynucleotides.

The isoprene synthase polynucleotide candidates were expressed in E. coli, isoprene prepared in a culture medium was measured to confirm an activity of the isoprene synthase, and the known isoprene synthase genes (P. alba, P. montana and A. hypogaea-derived isoprene synthase genes) were expressed in E. coli as a control group. Since cloning and culturing using cyanobacteria require quite a long time to work, they were conducted by selecting E. coli.

The genes for cloning were synthesized while removing a portion of encoding N-terminal sequence (N-terminal 40 aa of E. Pho and 48aa of I. bat) from the gene. In all genes, codon optimization was performed on Synechocystis sp. PCC6803 (SEQ ID NO: 3), and then codon optimization was performed on E. coli, by using GeneScript.

The fabricated construct was cloned in pBAT4 (Peranen J. et al, Anal. Biochem., 236:371-373, 1996) having trc promoter added thereto and cloned in pETDuet-1 (Novagen, USA, FIG. 6) having T7 promoter added thereto, so as to appropriate for expression in cyanobacteria, and when pBAT4 is used as a vector, the P. alba-derived polynucleotide and the I. batatas-derived polynucleotide were successfully cloned, and when pETDuet-1 is used as a vector, the I. batatas-, E. photiniifolius-, P. alba-, P. Montana- and A. hypogaea-derived isoprene synthase polynucleotides were cloned.

Example 3 Preparation of Isoprene in Recombinant Microorganism Containing Isoprene Synthase Gene Candidates

3-1: Transformation and Culturing

Plasmids containing isoprene synthase polynucleotide candidates cloned in Example 2 above were transformed into E. coli BL21, and expression of isoprene synthase was confirmed.

The selected transformants and parent strains were inoculated into an LB liquid medium containing 100 μg/mL ampicillin and cultured at 30° C. (or 37° C.) overnight, then the culture medium was diluted to 1:50, cultured until the OD600 at 30° C. is 0.6 to 0.7, and treated with IPTG so as to have a final concentration of 0.5 mM, thereby inducing an enzyme expression. The culturing was performed for 24 hours using a sealed 22 mL head-space bottle while inoculating the culture medium for 2 mL/bottle. At the same time, the culturing for protein analysis was performed using Erlenmeyer flask.

3-2: Detection and Quantification of Isoprene

Isoprene prepared in E. coli strain cloned with isoprene synthase was analyzed by solid-phase microexrction (SPME). An isoprene standard material containing 0.002% 4-tert-butylcatechcol as a stabilizer (Fluka nr529240 CAS 78-79-5) was used, and analyzed using divinylbenzene/carboxen/PDMS (DVB/CAR/PDMS) fiber (2 cm). CTC Combi PAL system (CTC Analytics AG, Switzerland) was used as a sampler, and the combination of Agilent 7890A gas chromatograph (GC, Agilent Technologies, USA) with a5975 C mass selective detector (MSD Agilent Technologies, USA) was used. In order to separate isoprene from ethanol which is volatile occurred during the culturing, HP-5, HP-35 (30 m) and BPX5 (60 m) columns were used, and isoprene was eluted by Lipodex (50 m) and HP-Innowax (60 m).

The elution time was 20 minutes, desorption time was 8 minutes, temperature in GC oven was between 40° C. (4 mins) and 70° C. (5° C./min), and the total running time was 16.3 mins. Temperature of the injector was 250° C., MS data were collected in m/z 35 to 350, and mass spectrum of isoprene was confirmed by comparison with NIST08 library.

The basic peak was m/z 67 and the mass peak was m/z 68, and other main fragments had m/z 53, 40 and 39. Calibration curves were measured by spike of isoprene in three different media (LB, BG11 and BG11 without Na₂CO₃). The medium had a concentration area of 2˜85 ng/2 mL, and the spike was performed on the sample with ethanol having the same amount (10 μl/bottle). The sample was put into a 22 mL head-space bottle used in both of the culturing and the analysis and was analyzed. The quantitative limit of the present invention was 0.5 ng/ml, and the detection limit was lower than the quantitative limit.

Expression in the pBAT4 constructs (trc promoter) of the P. alba- and I. batatas-derived isoprene synthase genes before and after the induction of the isoprene synthase was shown in Criterion TGX 4-20 gel with GelCode Blue dye (FIG. 7). Expression of the isoprene synthase and preparation of isoprene were confirmed by culturing five clones (A to E) of two constructs. After the induction, preparation of P. alba-derived isoprene synthase (64 kDa) was clearly confirmed, but I. batatas lysate (62 kDa) was not clearly confirmed due to high viscosity. After 24 hours of the induction, the isoprene prepared by the recombinant E. coli was analyzed by SPME method. As a result, it could be confirmed from FIG. 8 that the recombinant E. coli-derived sample containing l. batatas-derived isoprene synthase had particularly high isoprene productivity.

As a result of confirming expression of the isoprene synthase of the isoprene synthase clone of five kinds (I. batatas, E. photiniifolius, P. alba, P. montana and A. hypogaea) using pETDuet-1 vector, it could be confirmed from FIG. 9 that after 24 hours of the induction, all strains excluding E. photiniifolius showed clear isoprene synthase production.

As a result of analyzing the isoprene productivity in the five kinds of recombinant E. coli's after 24 hours of the induction, it could be confirmed from FIG. 10 that high isoprene productivity was shown in I. batatas- and P. alba-derived isoprene synthases, in particular, the highest isoprene productivity was shown in I. batatas-derived isoprene synthase, and in E. photiniifolius, an enzyme production was not confirmed in Criterion TGX 4-20 gel, but isoprene was prepared. The preparation of isoprene was not confirmed in A. hypogaea.

In addition, as a result of confirming growth of each recombinant E. coli strain with OD₆₀₀ values, it was confirmed that the growth of each strain was slightly inhibited by expression of the recombinant enzyme (FIG. 11).

Further, FIG. 12 shows time dependent isoprene productivity after expression of isoprene synthases in an E. coli strain transformed into a pETDuet-1 vector in which I. batatas of isoprene synthases and P. alba of isoprene synthases, respectably, are inserted. It could be confirmed that isoprene was accumulated steadily after expression of isoprene synthases and isoprene was also produced in an E. coli strain without inducing expression by using IPTG.

As a result of confirming time dependent growth of strain by using OD₆₀₀ value, after inducing expression by using IPTG in recombinant E. coli including IpS derived from I. batatas and recombinant E. coli including IpS derived from P. alba, growth of strain steadily decreases compared to strain without inducing treatment by using IPTG (FIG. 13).

Example 4 Synechocystis sp. Cells Expressing Isoprene Synthase

Synechocystis sp. PCC6803 is used as the parent strain and is referred to as the wild type (wt). The wt strain and transformants are maintained on BG-11 agar and in BG-11 liquid medium buffered with Hepes to pH 7.5 and is supplemented with 10 mM bicarbonate and with the antibiotics kanamycin, spectinomycin or chloramphenicol as appropriate. Instead of or in addition to bicarbonate, CO₂ enriched cultivation conditions are used to provide the carbon source. Synechocystis is cultivated at 30° C. under illumination (10-300 μmol photons m⁻² s⁻¹).

Synechocystis sp. is transformed by natural transformation using previously described methods. The presence of the transforming DNA is confirmed by PCR.

Constructs are designed for the expression of isoprene synthase in cyanobacteria. The isoprene synthase is expressed under the transcriptional control of an E. coli or a Synechocystis promoter, which allows expression of isoprene synthase in cyanobacteria. As an example, the E. coli trc or lac promoters or Synechocystis sp. psbA promoter and Synechocystis terminator or E. coli rrnB terminator or phage T7 terminator are used. The I. batatas isoprene synthase nucleotide sequence (SEQ ID: No. 2) was codon optimized for expression in Synechocystis sp. PCC6803 and synthesized by GenScript (Piscataway, N.J., USA). The expression cassettes for isoprene synthase and the antibiotic resistance marker are flanked by homologous sequences corresponding to a neutral site in the Synechocystis sp. genome to facilitate integration of the transforming DNA into the genome by homologous recombination.

Neutral sites e.g. between slr0168 and slr0338 in the Synechocystis sp. PCC6803 genome are suitable target loci for introducing the isoprene synthase. Other target sites can be chosen in order to knock out genes that potentially interfere with production of the desired product and its precursors. Genes required for biosynthesis of storage compounds such as glgA or glgC, involved in glycogen biosynthesis, are examples of such genes.

Example 5 Synechocystis sp. Cells Expressing One or More Enzymes of the Mevalonate Pathway

In combination with isoprene synthase expression other genes contributing to isoprene production are introduced into Synechocystis cells. For example, the idi gene coding for isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) is introduced to enhance conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) that is the substrate of isoprene synthase. Optionally one or more genes coding for the mevalonate pathway components, mevalonate kinase (EC2.7.1.36), phosphomevalonate kinase (EC2.7.4.2), pyrophoshomevalonate decarboxylase (EC 4.1.1.33), acetoacetyl-CoA thiolase (EC2.3.1.9), HMG-CoA synthase (EC2.3.3.10) or HMG-CoA reductase (EC1.1.1.34) are cloned under an E. coli or Synechocystis promoter that allows expression in cyanobacteria. Two or more genes are expressed as a transcriptional fusion under the control of under the transcriptional control of an E. coli or a Synechocystis promoter. Each open reading frame is preceded by a ribosome binding site (RBS) to allow independent translation of the polypeptides. The above mentioned polynucleotides and the antibiotic resistance marker are flanked by homologous sequences corresponding to the target locus of choice in the Synechocystis sp. genome to allow integration of the transforming construct into the genome.

Example 6 Synechocystis sp. Cells Over-Expressing One or More Polypeptides of the Non-Mevalonate Pathway

Although cyanobacteria possess the non-mevalonate pathway for synthesising isoprenoid precursors, overexpression of selected pathway components from homologous or heterologous sources will benefit isoprene production. Constructs are designed for the overexpression of the non-mevalonate pathway genes, deoxyxylulose-5-phosphate synthase (DXP synthase; EC2.2.1.7), DXP reductioisomerase (EC1.1.1.267), MEP-cytidyltransferase (EC2.7.7.60), CPD-ME-kinase (EC2.7.1.148), MECDP synthase (1.17.7.1), HMBDP synthase (EC1.17.7.1), HMBDP reductase (EC1.17.1.2), and isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) in Synechocystis sp. are designed using the same principle as described for the mevalonate pathway genes. One or more non-mevalonate pathway genes are introduced into Synechocystis sp. in combination with isoprene synthase expression.

Example 7 Production of Isoprene by Recombinant Synechocystis Cells Expressing Isoprene Synthase

Synechocystis transformants expressing isoprene synthase and optional other genetic modifications are tested for isoprene production using an in vivo assay. Wild type cells are studied in parallel as controls. As an example, cells are grown for 4 days at 30° C. with 100 rpm shaking under continuous illumination in BG11 liquid medium supplemented with bicarbonate as the carbon source and the appropriate antibiotics. Cells from 20 ml cultivation are harvested by centrifugation and suspended in 8 ml of BG11 medium supplemented with bicarbonate and antibiotics as appropriate in 20 ml GC-MS bottles which are sealed air-tight. When isoprene synthase is expressed under control of an IPTG inducible promoter 0.5 mM IPTG is added into to medium to induce isoprene production. Cultivations are also carried out without IPTG. The bottles are incubated at 30° C. with 100 rpm shaking for up to 240 h. Samples are taken at regular intervals during the cultivation. At each time point a bottle is removed, the cultivation is heated at 40° C. and isoprene is measured from the head space of the bottle using the GC-MS method described earlier for E. coli.

Isoprene production into the medium in aerobic cultures of Synechocystis is also analysed. The culture medium is sampled periodically and the samples are transferred into GC-MS vials, heated at 40° C., and isoprene is measured off-line from the head space of the bottle using the GC-MS method described earlier for E. coli.

Example 8 Production of Isoprene by Recombinant Synechocystis Cells in a Bioreactor

The inocula for bioreactor cultivation are grown at 30° C. with 100 rpm shaking under continuous illumination in BG11 liquid medium supplemented with bicarbonate as the carbon source and the appropriate antibiotics. The inoculum is transferred into BG11 medium in a photobioreactor. Bicarbonate or CO₂ gas is used as the carbon source. The photobioreactor is equipped with adjustable lightning and controllable gas inlet and outlet. The cells are grown at 30° C. and the cultivation is mixed with agitation or gas bubbling. Isoprene production is analysed off-line. The culture medium is sampled periodically and the samples are transferred into GC-MS vials, heated at 40° C., and isoprene is measured from the head space of the bottle using the GC-MS method described earlier for E. coli. Isoprene is harvested from the bioreactor flushing the reactor periodically with CO₂ as described (Bentley and Melis, 2012, Biotechnol Bioeng 109:100-109).

Example 9 Construction of Isoprene Producing Saccharomyces cerevisiae Strains

Constructs are designed for the expression of isoprene synthase in S. cerevisiae. The isoprene synthase nucleotide sequence is expressed from an autonomously replicating multi-copy vector containing a 2-micron replication origin or an ARS sequence, a centromeric vector, or integrated into the genome in one or more copies. Optional other genetic modifications such as overexpression of isopentenyl-diphosphate delta-isomerase (IDI) or mevalonate pathway or non-mevalonate pathway components are also introduced into S. cerevisiae. The constructs can be assembled by traditional cloning methods or by recombination cloning. For example, S. cerevisiae strain FY834 is used for recombination cloning (Winston, Dollard and Ricupero-Hovasse, 1995). S. cerevisiae strain H2798 (ura-) is the parental strain which is used for the production of ispS isoprene.

A vector, named SP17, containing three S. cerevisiae promoter-terminator pairs cloned into the pRS426 plasmid, is used. The truncated Hmg-CoA reductase (Polakowski et al. 1998, Appl Microbiol Biotechnol. 49:66-71) is amplified by PCR using the oligonucleotides

TAGCAATCTAATCTAAGTTTTAATTACAAACTCGAGTAAAATGGACCAATTGGTGAAAACTG (SEQ ID NO: 4) and CCAAACCTCTGGCGAAGAAGTCCAAAGCTGTCGACGGATTTAATGCAGGTGACGG (SEQ ID NO: 5) and cloned into a SP17 vector between e.g. the TEF1 promoter and ADH1 terminator by homologous recombination. The IspS nucleotide sequence of I. batatas (SEQ ID NO: 2) is codon optimized for expression in S. cerevisiae and synthesized by GenScript, amplified by PCR, and inserted between e.g. the PGK1 promoter and PGK1 terminator. The IDI nucleotide sequence is amplified by PCR and cloned between e.g. TPI1 promoter and CYC1 terminator by homologous recombination. Schematic representation of the construct is shown in FIG. 14. S. cerevisiae are transformed using the LiAc/PEG method (Gietz and Woods, 2002, Methods in Enzymology 350: 87-96). The resulting plasmid is transformed into S. cerevisiae strain and selected on synthetic complete medium lacking uracil (SCD-Ura). The presence of the transforming DNA is confirmed by PCR.

The S. cerevisiae IspS transformants are propagated aerobically at 30° C. with 250 rpm shaking in SC-Ura medium containing glucose, galactose or glycerol as the carbon source to produce the biomass for isoprene production. For isoprene production, aliquots of the precultures are transferred into fresh SC-Ura medium containing glucose, galactose or glycerol in tightly sealed flasks, which enable harvesting of isoprene. The bottles are incubated at 30° C. with 250 rpm shaking for up to 96 h. Samples are taken at regular intervals during the cultivation. At each time point a bottle is removed, the cultivation is heated at 40° C. and isoprene is measured from the head space of the bottle using the GC-MS method described earlier for E. coli.

Example 10 Construction of Pichia kudriavzevii Strains for Isoprene Production

Synthetic I. batatas IspS gene, optimized for expression in S. cerevisiae, was obtained from GenScript (Piscataway, N.J., USA). The IspS polynucleotide was introduced into P. kudriavzevii strain ATCC32196 under the PGK1 or TDH1 promoters of P. kudriavzevii together with the isopentenyl-diphosphate delta-isomerase (IDI) gene. The P. kudriavzevii PGK1 promoter and S. cerevisiae MEL5 terminator controlled the expression of the hygromycin resistance marker that was used for selection of transformants. The Pk promoters were amplified from the genomic DNA of P. kudriavzevii strain ATCC32196 essentially as described in US 2009/0226989 A1. Terminators from S. cerevisiae and P. kudriavzevii were used. The marker cassette was flanked by loxP sites to enable removal and re-use of the marker. The isoprene synthase and marker expression cassettes were flanked by homologous sequences corresponding to the P. kudriavzevii GPD1 locus to enable integration in the GPD1 locus. The resulting construct contained P.k. GPD1 5′ flanking region-P.k. PGK1 promoter-MEL5 or hygromycin marker-MEL5 terminator-P.k. promoter-isoprene synthase nucleotide sequence-P.k. or S.c. terminator-P.k. promoter-IDI-P.k. or S.c.-P.k. GPD1 3′ flanking region (FIG. 15).

A HMG-CoA reductase (Polakowski et al. 1998, Appl Microbiol Biotechnol. 49:66-71) was co-expressed with the isoprene synthase in P. kudriavzevii under control of the P. kudriavzevii promoter. The resulting construct contained P.k. PDC1 3′ flanking region-P.k. promoter-MEL5 or hygromycin marker-MEL5 terminator-P.k. promoter-HMG-CoA reductase nucleotide sequence-P.k. terminator-P.k. PDC1 5′ flanking region (FIG. 16).

The resulting constructs were transformed into P. kudriavzevii strain ATCC32196 using the LiAc/PEG method. The transformants were selected based on blue colour on yeast peptone dextrose (YPD) medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-alpha-gal) or for growth on melibiose, or for growth on YPD medium containing hygromycin essentially as described in US 2009/0226989 A1. The presence of the transforming DNA was confirmed by PCR. Transformants were cultured aerobically on synthetic complete medium or yeast peptone (YP) medium containing glucose, fructose or glycerol as the carbon source. For isoprene analysis, aliquots of the precultures are transferred into fresh medium in tightly sealed GC-MS bottles retaining isoprene. The bottles were incubated at 30° C. with 100 rpm shaking for up to 96 h. Samples were taken at regular intervals during the cultivation. At each time point a bottle was removed, the cultivation was heated at 40° C. and isoprene was measured from the head space of the bottle using the GC-MS method described earlier for E. coli except that the medium used for the calibration curve was YP-medium containing 0.5% ethanol.

The expression vector for an N-terminally truncated S. cerevisiae HMG1 was named pPK-HMG (FIG. 16, SEQ ID NO: 6) and targeted for integration into the P. kudriavzevii PDC1 locus. The N-terminally truncated HMG1 gene (SEQ ID NO: 34) was amplified from genomic DNA of S. cerevisiae W303 by PCR using primers (5′-AGCGCGGATCCATGGACCAATTGGTGAAAACTGAAG) (SEQ ID NO: 7) and (5′-AGCGCGGATCCCACATGGTGCTGTTGTGCTTC) (SEQ ID NO: 8) and expressed under the control of the P. kudriavzevii PGK1 promoter and S. cerevisiae ADH1 terminator. The expression construct with the MEL5 marker and P. kudriavzevii PDC1 homology regions was released with NotI from plasmid pPK-HMG and transformed into P. kudriavzevii VTT-C-79090T (ATCC32196) using the lithium acetate method and colonies were screened based on blue colour on YPD-agar containing 40 μg/ml 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. A transformant Pk/HMG1 was selected for continuation.

The vector for expression of I. batatas IspS and S. cerevisiae IDI1 was named pPK-IspS-IDI (FIG. 15; SEQ ID NO: 9). The integration was targeted to the P. kudriavzevii GPD1 locus. The P. kudriavzevii GPD1 homology regions were amplified by PCR from genomic DNA of P. kudriavzevii VTT-C-79090T (ATCC32196). The I. batatas IspS gene with a C-terminal StrepII-tag was codon optimized for expression in S. cerevisiae and synthesized by Genscript (Hongkong) (SEQ ID NO: 33). I. batatas IspS was expressed under control of the P. kudriavzevii PGK1 promoter and S. cerevisiae ADH1 terminator. The IDI1 gene (SEQ ID NO: 35) was amplified by PCR from genomic S. cerevisiae DNA using primers 918IDISc-F (5′-CTTTTTACAACAAATATAAAACCAAAAGCGGCCGCTTAATTAAAAAATGACTGCCGACAACAATAGTATG) (SEQ ID NO: 10) and C031_919new_IDISc_R (5′-AGAGACATGGGAGATCCCGCGGGCGGCCGCTTAATTAATTATAGCATTCTATGAATTTGCC) (SEQ ID NO:11), and placed under the control of the P. kudriavzevii TDH1 promoter and S. cerevisiae PGK1 terminator. The expression construct with a hygromycin resistance marker and P. kudriavzevii GPD1 homology regions was released with NotI from plasmid pPK-IspS-IDI and transformed into P. kudriavzevii VTT-C-79090T (ATCC32196) and into P. kudriavzevii Pk/HMG1 using the lithium acetate method (Gietz et al. 1992, Nucleic Acids Res 20:1425-1425) and colonies were selected on YPD-agar containing 450 μg/ml hygromycin B as the selective agent. Transformed colonies Pk/IspS+IDI1-69, Pk/IspS+ID1I-72, Pk/ISpS+IDI1-74 and Pk/IspS+IDI1+HMG1-79 were isolated and tested for isoprene production.

P. kudriavzevii VTT-C-79090T (ATCC32196) and the Pk/HMG1, Pk/IspS+IDI1 and Pk/IspS+IDI1+HMG1 transformants were grown in YPD medium o/n at 30° C. Cells were collected by centrifugation and suspended in YP−1% glucose or YP+1% glycerol to an OD₆₀₀=3.2 ml aliquots were sealed in 22 ml headspace bottles and incubated o/n at 30 with 100 rpm shaking. Isoprene production was measured as described above except that the isoprene standards were prepared in YP-medium containing 0.5% ethanol. Isoprene concentrations measured are shown in FIG. 18. FIG. 18. shows isoprene production by P. kudriavzevii transformants in YP medium containing 1% glucose (grey) or 1% glycerol (black) as the carbon source. All transformants expressing IspS and IDI1 produced isoprene while the parent strain and the strain expressing only HMG1 did not produce detectable isoprene. The amount of isoprene measured ranged from 1 to 12 μg in the 2 ml samples depending on the strain and cultivation conditions. More isoprene was produced on YP-glycerol medium than on YP-glucose medium. The presence of HMG1 in addition to IspS and IDI1 increased isoprene production approximately 3-fold in YP-glycerol medium and 2-fold in YP-glucose medium.

Example 11 Construction of Trichoderma reesei Strains Overexpressing an Isoprene Synthase for Isoprene Production I

Synthetic I. batatas IspS gene, optimized for expression in T. reesei, is obtained from GenScript (Piscataway, N.J., USA) (SEQ ID NO: 36). The codon optimized IspS polynucleotide was introduced into T. reesei strain Rut-30 under the control of gpdA promoter of Aspergillus nidulans or under the control of the cbh1 promoter of T. reesei.

The resulting constructs contained the T. reesei cbh1 5′ flank region-cbh1 promoter-isoprene synthase-nucleotide sequence A. nidulans gpdA promoter hygromycin resistance marker (hph) and A. nidulans trpC terminator-T. reesei cbh1 terminator and 3′ flank region. The resulting constructs were transformed into T. reesei using the PEG-mediated protoplast transformation. The transformants were selected based on hygromycin resistance. Instead of hph, the acetamidase A. nidulans amdS gene can be used as the selective marker and the transformants were selected based on the ability to use acetamide as the sole nitrogen source. Alternatively, the isoprene synthase was expressed under the A. nidulans gpdA promoter. The codon optimized isoprene synthase polynucleotide was cloned into an expression vector containing the A. nidulans gpdA promoter-isoprene synthase-nucleotide sequence-A. nidulans trpC terminator-A. nidulans gpdA promoter hygromycin resistance marker (hph) and A. nidulans trpC terminator. Schematic view of the plasmid used for transformation was shown in FIG. 16. The transformants were purified from colonies originating from single spores, and the integration of the expression cassette into the genome was confirmed by PCR amplification of the expression cassette. The polynucleotides were integrated into the cbh1 locus or into a non-homologous site in T. reesei genome.

T. reesei transformants containing the ispS polynucleotide were cultivated in shake flasks in medium containing 7.6 g/L (NH₄)₂SO₄ 15.0 g/L KH₂PO₄, 2.4 mM MgSO.7H₂O, 4.1 mM CaCl₂.H₂0, 3.7 mg/L CoCl₂, 5 mg/L FeSO_(4.)7H₂0, 1.4 mg/L ZnSO₄.7H₂O, 1.6 mg/L MnSO₄.7H₂O, and a 10 g/L carbon source or a mixture of carbon sources selected from cellulose, xylan, glucose, lactose, glycerol, galactose, sorbose, sorbitol, xylose, arabinose or 0.7 mM sophorose. The pH of the medium was adjusted to 4.8 by addition of KOH. The cultures were inoculated with 8×10⁷ spores/200 ml medium and grown for up to 7 days in conical flasks at 28° C. with shaking at 250 rpm. The culture medium was sampled periodically and the samples were transferred into GC-MS vials, heated at 40° C., and isoprene was measured off-line from the head space of the bottle using the GC-MS method described earlier for E. coli.

Alternatively, the aliquots of the culture transferred into fresh medium in tightly sealed bottles. The bottles were incubated at 28° C. with 250 rpm shaking for up to 96 h. Samples were taken at regular intervals during the cultivation. At each time point a bottle was removed, the cultivation was heated at 40° C. and isoprene was measured from the head space of the bottle using the GC-MS method described earlier for E. coli.

Example 12 Construction of Trichoderma reesei Strains Overexpressing an Isoprene Synthase for Isoprene Production II

Strains

Escherichia coli DH5α (Life Technologies) was used for propagation of the plasmids. Trichoderma reesei QM6a (VTT-D-071262T, ATCC13631), RutC-30 (VTT-D-086271, ATCC56765), and M122 (RutC-30 mus53Δ) were obtained from VTT Culture Collection (Espoo, Finland). Spore suspensions were prepared by cultivating the fungus on potato-dextrose plates (BD, Sparks, Md., USA) for 5-7 days, after which the spores were harvested, suspended in a buffer containing 0.8% NaCl, 0.025% Tween-20 and 20% glycerol, filtered through cotton and stored at −80° C.

Expression Vector

The vector for expression of I. batatas IspS in T. reesei was named pCIL-105 (FIG. 19 SEQ ID NO:12). The backbone for the expression vector contained 5′ and 3′ flank regions (<1000 bp) of the T. reesei pep4 (tre77579) gene in pRS426 (ATCC77107) vector background. The primer sequences indicating the start and end regions for both pep4 flanks are shown in Table 1. The selection marker (pyr4) in the vector backbone pCIL102 was removed by NotI digestion and replaced by hygromycin resistance cassette obtained from plasmid pCIL-107 by NotI digestion. The gene encoding I. batatas isoprene synthase with a C-terminal StrepII-tag was obtained from E. coli expression plasmid pCIL41 by NdeI-AvrII digestion. The Aspergillus nidulans gpdA promoter, A. nidulans trpC terminator and a bridge fragment (part of pep4 3′flank) were produced by PCR using primers in Table 1 and plasmid pCIL104 containing these elements as templates. PCR amplification was carried out with KAPA HiFi HotStart ReadyMix PCR kit (KAPA Biosystems). All fragments were separated with agarose gel electrophoresis and isolated with a gel extraction kit (Qiagen). The PCR fragments contained 30 bp or 40 bp overlapping sequences needed for cloning the expression construct using homologous recombination system in yeast. The vector backbone and the appropriate digestion and PCR fragments were transformed into S. cerevisiae (strain H3488/FY834) according to Gietz, R. D., and Woods, R. A. (2002, Guide to Yeast Genetics and Molecular and Cell Biology, Pt B 350, pp. 87-96). The plasmid DNA from the yeast transformants was rescued by transformation into E. coli and checked from a few clones by digestion. The clone taken further was verified by sequencing and named pCIL105 (SEQ ID NO: 12).

Strain Generation

For T. reesei transformation, the expression construct was released from pCIL105 with MssI and the correct fragment was purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). T. reesei strain RutC-30 or T. reesei strain M122 (RutC-30 Δmus53) was transformed with 5 μg of expression cassette fragment according to Penttila et al. 1987 (Gene 61: 155-164). Transformants were selected on Trichoderma minimal medium containing 125 μg/ml hygromycin B (Calbiochem). The transformants were streaked onto Trichoderma minimal medium agar with 0.1% (v/v) Triton X-100 and 150 μg/ml Hygromycin B and cultivated at +28° C. Transformants growing on selective plates were screened by PCR for correct 5′ and 3′integration or for the presence of the IspS expression cassette (FIG. 20) somewhere else in the genome using the primers shown in Table 2. FIG. 20. shows PCR analysis for the presence of the IspS expression cassette in T. reesei transformed with pCIL105. Wild type strain QM6a, parent and DDIW are negative controls. For PCR, internal primers (SEQ ID NO: 31 and SEQ ID: NO 32) shown in Table 2 were used. The expected product size was approximately 1.1 kb.

TABLE 1 Primers used to generate fragments for cloning the expression vector pCIL105 for I. batatas IspS-C-StrepII. Primer Sequence Product 77579 5f TCAGGTCAACCACCGAGGAC (SEQ ID NO: 13) pep4 5′flank 77579_5r TGAATGGGATGGTTCGATTG (SEQ ID NO: 14) 77579_3f AGGTAGACGCTTTGCGAGTG (SEQ ID NO: 15) pep4 3′flank 77579_3r TGAACTGACGCGGACTGA (SEQ ID NO: 16) C017_gpdA_rec_for CCTCTGGCAGCAATCGAACCATCCCATTCATTAATTAA gdpA prom GCTCCTTATTGAAGTCGGAGG (SEQ ID NO: 17) C018_gpdA_rec_rev ATGAGGAGGGTTGATAGTTTGCTGAGCGGCGGGCAGT CATGATGTCTGCTCAAGCGGG (SEQ ID NO: 18) C019_trpC_rec_for TAATCCAGTGGAATCTGTCCCACCCACAATTTGAAAAG trpC term TAAGATCCACTTAACGTTACTGAAATCA (SEQ ID NO: 19) C020_trpC_rec_rev AAGGGGACCGGCCGCTAGTCTCACCGTTATGCGGCCG CTTAATTAAGAGTGGAGATGTGGAGTGGG (SEQ ID NO: 20) C021_pep4_3frec_for GATAACCCATCGGCAGCAGATGATAATGATTCCGCAG pep4 bridge CACGCGGCCGCAGGTAGACGCTT (SEQ ID NO: 21) C022_pep4_3f_rev GAGGTGCCAAAGCCGTTTCG (SEQ ID NO: 22)

TABLE 2 Primers used to screen for correct integration of the expression cassette or the presence of the IspS cassette in the T. reesei genome. Primer Sequence Product T302_77579_5int GATTCATCACAGGGGCAGTC (SEQ ID NO: 23) 5′integration T624_gpdA_seqR1 CTCCATATTCTCCGATGATGC (SEQ ID NO: 24) T302_77579_5int GATTCATCACAGGGGCAGTC (SEQ ID NO: 25) 5′integration C046_gpdA_rev TATCCTCTTGACACCGCTCC (SEQ ID NO: 26) T415_77579_3screen ACGCCGTTGCTGAGCCTTG (SEQ ID NO: 27) 3′integration T1411_cbh2t_end_f CCAATAGCCCGGTGATAGTC (SEQ ID NO: 28) T415_77579_3screen ACGCCGTTGCTGAGCCTTG (SEQ ID NO: 29) 3′integration T1404_cbh2term_for CCGTCTAGCGCTGTTGATTG (SEQ ID NO: 30) C009_Ibat_for1 GATCAACTTAGCACGCATTG (SEQ ID NO: 31) internal C029_PKIp_rev TTTGCTCCAACTCAGGCG (SEQ ID NO: 32)

Example 13 Construction of Aspergillus niger Strains Overexpressing an Isoprene Synthase for Isoprene Production

The codon optimized isoprene synthase polynucleotide of example 3 was cloned into an expression vector containing the A. nidulans gpdA promoter-isoprene synthase polynucleotide-A. nidulans trpC terminator-A. nidulans gpdA promoter hygromycin resistance marker (hph) and A. nidulans trpC terminator (FIG. 17). The resulting construct was transformed into A. niger. Transformation was performed using the PEG-mediated protoplast transformation. The transformants were selected based on hygromycin resistance. The presence of the transforming DNA was confirmed by PCR.

A. niger was grown in the production medium (The defined medium of Vogel described by Mojzita et al., 2010, with 20 g/L glucose or xylose as a carbon source). Pre-cultures were grown in the medium containing 10 g/L yeast extract, 20 g/L peptone and 30 g/L gelatine (50 ml medium in 250 ml flasks). Mycelium from 50 ml cultures was collected by filtration, washed with sterile H₂O and re-suspended in 20 ml of the production medium in 250 ml flasks. Cultures were incubated at 30° C., 250 rpm. The culture medium was sampled periodically and the samples were transferred into GC-MS vials, heated at 40° C., and isoprene was measured off-line from the head space of the bottle using the GC-MS method described earlier for E. coli. Alternatively, for isoprene production, aliquots of the culture were transferred into fresh medium in tightly sealed GC-MS bottles. The bottles were incubated at 30° C. with 250 rpm shaking for up to 96 h. Samples were taken at regular intervals during the cultivation. At each time point a bottle was removed, the cultivation was heated at 40° C. and isoprene was measured from the head space of the bottle using the GC-MS method described earlier for E. coli.

Example 14 Ipomoea batatas IspS Expression in Plant Cell Cultures

The I. batatas isoprene synthase polynucleotide was cloned into an over expression vector functional in various plant species under the control of the CaMV35S promoter for expression in plant cells. The expression vector had the hygromycin or kanamycin resistance marker for the selection of transformants. Nicotiana tabacum bright yellow (BY-2) cell suspension cultures or hairy root cultures were maintained and transformed with the isoprene synthase expression construct essentially as described in (Hakkinen et al. 2007 Phytochemistry 68:2773-2785). Transformants were selected based on antibiotic resistance. The presence of the transforming DNA was confirmed by PCR. For isoprene accumulation analysis, hairy roots were incubated in 20 ml medium in 100 ml shake flasks and cultivated in a rotary shaker (70 rpm, 24° C.) in modified Gamborg B5 medium without casein (Jouhikainen et al., Planta, 208:545-551, 1999) for up to 28 days. The culture medium was sampled periodically and the samples were transferred into GC-MS vials, heated at 40° C., and isoprene was measured off-line from the head space of the bottle using the GC-MS method described earlier for E. coli.

Although specific embodiments of the present invention are described in detail, it will be apparent to those skilled in the art that the specific description is merely desirable exemplary embodiment and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalent thereof.

Sequence Listing Free Text

Attach the electronic file. 

The invention claimed is:
 1. A recombinant vector comprising a codon-optimized polynucleotide encoding an isoprene synthase operably introduced therein, wherein the codon-optimized polynucleotide has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO:
 33. 2. The recombinant vector of claim 1, wherein the recombinant vector comprises a promoter for expressing said polynucleotide.
 3. The recombinant vector of claim 2, wherein the promoter is selected from the group consisting of psbA2, trc, rbcL, petJ, psaA, psaB, tac, cpcB, petC and lac for expressing in cyanobacteria, wherein the promoter is selected from the group consisting of PGK1, TPI1, TDH, PDC1, FBA1, ENO1, ENO2, PYK1, ADH1, and TEF1 for expressing in yeast, wherein the promoter is selected from the group consisting of cbhl, gpdA, glaA, pdc and exlA for expressing in filamentous fungi, or wherein the promoter is CMV35S for expressing in plant cells.
 4. The recombinant vector of claim 1, wherein the codon-optimized polynucleotide has the nucleotide sequence of SEQ ID NO:
 3. 5. The recombinant vector of claim 1, wherein the codon-optimized polynucleotide has the nucleotide sequence of SEQ ID NO:
 33. 6. A recombinant host cell comprising a recombinant vector comprising a codon-optimized polynucleotide encoding an isoprene synthase operably introduced therein, wherein the codon-optimized polynucleotide has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO:
 33. 7. The recombinant host cell of claim 6, wherein the host cell is selected from the group consisting of cyanobacteria, yeast, filamentous fungi and plant cell.
 8. The recombinant host cell of claim 7, wherein the cyanobacterium is unicellular cyanobacterium or multicellular cyanobacterium.
 9. The recombinant host cell of claim 8, wherein the unicellular cyanobacterium is Synechocystis sp. or Synechococcus sp.
 10. The recombinant host cell of claim 8, wherein the multicellular cyanobacterium is Gloeocapsa sp. or filamentous cyanobacterium sp.
 11. The recombinant host cell of claim 10, wherein the filamentous cyanobacterium is selected from the group consisting of Nostoc sp., Anabaena sp., and Arthrospira sp.
 12. The recombinant host cell of claim 7, wherein the yeast is selected from the group consisting of Saccharomyces sp., Pichia sp., Candida sp., Kazachstania sp., Kluyveromyces sp., Hansenula sp., Rhodosporidium sp., Cryptococcus sp., and Yarrowia sp.
 13. The recombinant host cell of claim 7, wherein the filamentous fungus is selected from the group consisting of Trichoderma sp., Mucor sp., Mortierella sp., Neurospora sp., and Aspergillus sp.
 14. The recombinant host cell of claim 7, wherein the plant cell is selected from the group consisting of Nicotiana sp., Catharantus sp., and Hyoscyamus sp.
 15. The recombinant host cell of claim 6, further comprising one or more genes, which are introduced thereto, selected from the group of: (i) genes coding for the mevalonate pathway components, selected from the group of gene coding for mevalonate kinase (EC2.7.1.36), phosphomevalonate kinase (EC2.7.4.2), pyrophoshomevalonate decarboxylase (EC 4.1.1.33), acetoacetyl-CoA thiolase (EC2.3.1.9), HMG-CoA synthase (EC2.3.3.10), and HMG-CoA reductase (EC1.1.1.34); and (ii) genes coding for isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2).
 16. The recombinant host cell of claim 6, further comprising a gene which is introduced thereto, wherein the gene is an idi gene coding for isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) and/or a gene coding for HMG-CoA reductase (EC1.1.1.34).
 17. A method for preparing isoprene comprising: (a) culturing the recombinant host cell of claim 6, thereby producing isoprene; and (b) recovering the isoprene.
 18. The method of claim 17, wherein the culturing of step (a) is performed on a medium comprising a carbon source selected from the group consisting of CO₂, bicarbonate, glucose, glycerol, glycerin, dihydroxyacetone, yeast extract, biomass, molasses, sucrose, galactose, sorbose, sorbitol, xylose, arabinose, cellulose, xylan, lactose and oil. 